Hepatocytes constitute approximately 80% of the cells in the liver and are critical for both the activation and eventual detoxification of many pharmacological compounds, toxins or xenobiotics. Increased demand for and availability of new drugs, as well as stricter regulatory and safety testing prior to market approval, have made isolated primary hepatocytes an invaluable resource for studying drug metabolism, efficacy and toxicity in a laboratory setting.
In recent years, significant advancements have been made in the isolation and cryopreservation of primary donor hepatocytes, which can be rapidly thawed and immediately used for experimentation. However, studying hepatic metabolism in hepatocytes isolated from one human liver (individual donor) does not accurately reflect liver function in the overall population since variations in gender, age, ethnicity, health status, genetic background, and other factors skew test results. A more accurate measure of hepatic metabolism is to a use a mixture or “pool” of individual donor cells to create a heterogeneous population of hepatocytes.
A number of methods have been proposed for pooling hepatocytes. These protocols often employ lengthy procedures in which cells are exposed to both physical and chemical stress that reduce the total number of viable cells. For example, the method in U.S. Pat. No. 7,604,929 utilizes a density gradient centrifugation step before the second or final cryopreservation step. This subjects the cells to chemical and mechanical stress that either results in cell loss (FIGS. 1A-B) or weakens the cells such that they die during cryopreservation. Another method disclosed in WO 2014/045202 A2 maintains the hepatocytes in a cryopreservative solution throughout the pooling process. Cryopreservative solutions contain toxic reagents, such as dimethyl sulfoxide (DMSO), which are known to cause cell death.
The proposed system and method seeks to employ techniques to reduce cell loss and therefore increase the total number of viable cells throughout the process.